Abstract: Objective　To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Methods　Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow⁃derived macrophages (BMDMs) with mouse macrophage colony⁃stimulating factor (M⁃CSF), which served as controls (BMDMs⁃M0). BMDMs⁃M0 induced M2 macrophages by interleukin⁃4 for 24 hours served as the IL⁃4 induction group, and BMDMs⁃M0 co⁃cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM⁃CF co⁃culture group, while BMDMs⁃M0 co⁃cultured with E. multilocularis protoscolex (PSC) at a ratio of 500∶1 served as the BMDM⁃PSC co⁃culture group. The types of polarization of BMDMs co⁃cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism⁃related enzymes was quantified using fluorescent quantitative real⁃time PCR (qPCR) and Western blotting assays. Results　There were significant differences among the four groups in terms of Arginase⁃1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages⁃derived C⁃C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin⁃like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde⁃3⁃phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL⁃6 (F = 1 823.00, P < 0.000 1), IL⁃10 (F = 291.70, P < 0.000 1), IL⁃1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)⁃α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)⁃β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM⁃PSC co⁃culture group [(22.87% ± 1.48%) vs. (1.70% ± 0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM⁃CF co⁃culture group [(20.07% ± 0.64%) vs. (1.93% ± 0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM⁃CF and BMDM⁃PSC co⁃culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL⁃10, IL⁃1β, TNF⁃α and TGF⁃β in the BMDM⁃CF and BMDM⁃PSC co⁃culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM⁃PSC co⁃culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL⁃6 mRNA expression in the BMDM⁃CF co⁃culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL⁃6 and TNF⁃α and higher TGF⁃β mRNA expression (both P values < 0.05) was detected in the IL⁃4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL⁃4 induction group and BMDM⁃PSC co⁃culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM⁃PSC co⁃culture group and a lower ECAR was found in the IL⁃4 induction group than in the control group (both P values < 0.05). Conclusions Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high⁃energy and high⁃glycolytic metabolism, and affects inflammatory responses in BMDM.

Key words: Echinococcus multilocularis, Hydatid fluid, Protoscolex, Macrophage polarization, Glucose metabolism, Metabolic phenotype

摘要： 目的　探讨多房棘球蚴对巨噬细胞糖代谢表型转变、极化类型及炎症反应的影响，为多房棘球蚴病发病机制研究提供参考。方法　分离6～8周龄C57BL/6J小鼠骨髓细胞，用小鼠巨噬细胞集落刺激因子（macrophage colony⁃stimulating factor，M⁃CSF）诱导成骨髓巨噬细胞（bone marrow⁃derived macrophage，BMDM）⁃M0（对照组）；以BMDM⁃M0经白细胞介素（interleukin）⁃4诱导24 h后的M2型巨噬细胞作为IL⁃4诱导组，以BMDM⁃M0与2.4 ng/mL多房棘球蚴囊液（cystic fluid，CF，）共培养的细胞作为BMDM与多房棘球蚴CF共培养组，以BMDM⁃M0与多房棘球蚴原头节（protoscolex，PSC）按500∶1共培养的细胞作为BMDM与PSC共培养组。采用流式细胞术分析与多房棘球蚴CF、PSC共培养后的巨噬细胞极化类型，用实时荧光定量PCR（fluorescent quantitative real⁃time PCR， qPCR）、Western blotting分析其标志物、炎症因子、糖代谢相关酶的表达水平，并通过细胞能量代谢分析各组细胞葡萄糖代谢表型。结果　4组细胞精氨酸合酶1（arginase⁃1，Arg1）（F = 1 457.00，P ＜ 0.000 1）、巨噬细胞衍生趋化因子22（macrophages⁃derived C⁃C motif chemokine 22，Ccl22）（F = 22 203.00，P ＜ 0.000 1）、抵抗素样α（resistin⁃like α，Retnla）（F = 151.90，P ＜ 0.000 1）、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）（F = 107.80，P ＜ 0.001）、己糖激酶（hexokinase，HK）（F = 9 389，P ＜ 0.000 1）、丙酮酸激酶（pyruvate kinase，PK）（F = 641.40，P ＜ 0.000 1）、磷酸果糖激酶1（phosphofructokinase1，PFK1）（F = 43.97，P ＜ 0.01）、葡萄糖激酶（glucokinase，GK）（F = 432.50，P ＜ 0.000 1）、丙酮酸脱氢酶激酶（pyruvate dehydrogenase kinases，PDK1）（F = 737.30，P ＜ 0.000 1）、乳酸脱氢酶（lactic dehydrogenase，LDH）（F = 3 632.00，P ＜ 0.000 1）、葡萄糖转运体1（glucose transporter 1，GLUT1）（F = 532.40，P ＜ 0.000 1）、甘油醛⁃3⁃磷酸脱氢酶（glyceraldehyde⁃3⁃phosphate dehydrogenase，GAPDH）（F = 460.00，P ＜ 0.000 1）、柠檬酸合酶（citrate synthase，CS）（F = 5 642.00，P ＜ 0.01）、糖原合成酶1（glycogen synthase 1，GYS1）（F = 273.30，P ＜ 0.000 1）、IL⁃6（F = 1 823，P ＜ 0.000 1）、IL⁃10（F = 291.70，P ＜ 0.000 1）、IL⁃1β（F = 986.60，P ＜ 0.000 1）、肿瘤坏死因子（tumor necrosis factor，TNF）⁃α（F = 334.80，P ＜ 0.000 1）、转化生子因子（transforming growth factor，TGF）⁃β（F = 163.30，P ＜ 0.001）mRNA表达水平差异均有统计学意义。BMDM与多房棘球蚴PSC共培养组M2型巨噬细胞比例（22.87% ± 1.48%）高于M1型巨噬细胞（1.70% ± 0.17%）（t = 24.61，P ＜ 0.001），BMDM与多房棘球蚴CF共培养组M2型巨噬细胞比例（20.07% ± 0.64%）高于M1型巨噬细胞比例（1.93% ± 0.25%）（t = 45.73，P ＜ 0.001）。与对照组相比，BMDM与多房棘球蚴CF共培养组、BMDM与多房棘球蚴PSC共培养组M2型巨噬细胞标志物Arg1、Ccl22、Retnla mRNA表达水平均升高（P均 ＜ 0.01），而M1型巨噬细胞标志物iNOS mRNA表达水平无明显变化（P ＞ 0.05），糖酵解关键酶HK、PK、PFK mRNA表达水平升高（P均 ＜ 0.01），炎症因子IL⁃10、IL⁃1β、TNF⁃α、TGF⁃β mRNA表达水平升高（P均 ＜ 0.01）。此外，与对照组相比，BMDM与多房棘球蚴PSC共培养组HK、PK、PFK蛋白表达水平，GLUT1、GAPDH、IL⁃6 mRNA表达水平均升高（P均 ＜ 0.05）；BMDM与多房棘球蚴CF共培养组HK蛋白表达水平升高（P＜ 0.05）；IL⁃4诱导组HK、PK、PFK蛋白及mRNA表达水平均升高（P均 ＜ 0.01），IL⁃6、TNF⁃α mRNA表达水平均降低（P均 ＜ 0.05），TGF⁃β mRNA表达水平升高（P ＜ 0.01）。糖酵解压力测试显示，对照组、IL⁃4诱导组、BMDM与多房棘球蚴PSC共培养组细胞外酸化率（extra cellular acidification rate，ECAR）差异有统计学意义（F = 124.4，P ＜ 0.05）；与对照组相比，BMDM与多房棘球蚴PSC共培养组ECAR增高，IL⁃4诱导组ECAR降低（P均 ＜ 0.05）。结论　多房棘球蚴CF/PSC处理使BMDM主要向M2型极化，糖代谢向高能量、高糖酵解代谢表型转变，并影响其炎症反应。

关键词: 多房棘球蚴, 囊液, 原头节, 巨噬细胞极化, 葡萄糖代谢, 代谢表型